Professor Don Grierson

Research and Interests

My research is aimed at identifying and characterising genes
that are involved in ethylene signalling and ripening in tomato and understanding
how ethylene signalling interacts with regulatory networks and other signalling
pathways to control flower and fruit development and ripening. A summary of
recent and current work is given in the Abstract below of a research presentation
at a recent conference.
Previously I was PVC for Research for the University of Nottingham from 2003-2007.
During this time I was responsible for the University Research Strategy and
the University achieved record research grant totals in four successive years
(2004-2007). I was also responsible for the preparations for, and submission
of, the University returns in 2007 for the 2008 Research Assessment Exercise.
Independent analysis published by “Research Fortnight” showed the
University had moved from 14th to 7th position in the national rankings for
“Research Power” when the results were published in 2008. Furthermore,
Nottingham topped the table as the University likely to receive the largest
increase in research funding from the national funding agency (HEFCE) as a result
of the RAE assessment (Research Fortnight 4/2/2009). Previously I was Head of
the Department of Physiology and Environmental Science at the Sutton Bonington
Campus (1988-1991 and 1992-1994), Head of the Plant Sciences Division from 1997-
2002, and Foundation Head of the School of Biosciences from 2000-2002.

Ethylene, synthesised via the Yang Cycle, affects many aspects of plant development,
including vegetative growth, ripening, senescence, abscission, and responses
to biological and environmental stresses. Tomato has become the model for climacteric
fruit ripening (Alexander and Grierson, 2002), and inhibition of the ethylene
biosynthesis genes ACC synthase (ACS) and ACC oxidase (ACO) delays or prevents
ripening. After perception, ethylene stimulates a signalling cascade, which
has been studied largely in Arabidopsis, which leads to a series of changes
in expression of genes encoding proteins that catalyse alterations in colour,
texture, flavour, aroma, and nutrient content, to produce fruit attractive to
a consumer.
Plants have multiple genes for both ACS and ACO, which are differentially expressed
during development, but very little is known about the regulation of ethylene
synthesis at different stages of the life cycle. We have shown by gel retardation
assay that LeHB-1, a previously uncharacterized tomato homeobox protein, interacts
with the promoter of LeACO1, an ACC oxidase gene expressed during ripening.
Inhibition of LeHB-1 accumulation in tomato fruit using virus-induced gene silencing
greatly reduced LeACO1 mRNA levels and inhibited ripening. Conversely, ectopic
overexpression of LeHB-1 by viral delivery to developing flowers elsewhere on
injected plants triggered altered floral organ morphology, including production
of multiple flowers within one sepal whorl, fusion of sepals and petals, and
conversion of sepals into carpel-like structures that grew into fruits and ripened.
This suggests the LeHB-1 regulatory network plays a role in flower and fruit
development as well as ripening (see Lin et al., 2008b). Interestingly, LeACS2
gene expression is also regulated by a homeotic protein LeMADS-RIN (Ito et al,
2008).
Components of the ethylene signalling network found in Arabidopsis are conserved
in tomato, and both the ethylene receptors and CTR1-like proteins in tomato
are encoded by multi-gene families. As in Arabidopsis, control of ethylene responses
operates by a receptor-inhibition mechanism in tomato. Inhibition is relieved
by ethylene binding to the receptor, enabling the ripening cascade to be initiated.
This has been confirmed by showing that Nr tomatoes expressing the mutant NR
ethylene receptor, which cannot bind ethylene and therefore prevents normal
ripening, can be restored to partial or complete ripening by antisense inhibition
of production of the mutant mRNA (Hackett et al 2000). Although it was initially
thought that the ethylene receptors might be functionally equivalent, we now
know that different receptors have particular roles at specific stages of the
life cycle and it has been proposed that in tomato the ethylene receptors NR,
LeETR4 & 6 have unique roles (Kevany et al, 2007).
Although it has been shown that tomato CTR1, 3 & 4 can partially or completely
complement the Arabidopsis ctr1 mutant (Adams-Phillips et al, 2004), it was
not known which receptors bound to these multiple CTRs in tomato. We have shown,
using yeast two-hybrid assay, that the tomato receptors LeETR1, LeETR2 and NEVER-RIPE
(NR) can interact with multiple LeCTRs. In vivo protein localization studies
using fluorescent proteins revealed that the ethylene receptor NR was targeted
to the endoplasmic reticulum (ER) when transiently expressed in onion epidermal
cells, whereas the four CTR proteins alone were found in the cytoplasm and nucleus.
When co-expressed with NR, three LeCTRs (CTR1, 3 and 4), but not CTR2, also
adopted the same ER localization pattern in an NR receptor-dependent manner
but not in the absence of NR. The receptor-CTR interactions were confirmed by
bimolecular fluorescence complementation (BiFC) showing that NR could form a
protein complex with CTR1, 3 and 4 through direct protein-protein interaction
at the ER (see Zhong et al., 2008). Arabidopsis transcription factor EIN3 operates
downstream of CTR1. In tomato there are multiple EIN3-like (EIL) proteins and
it has been suggested that these are functionally redundant (Tieman et al, 2001).
We have shown, however, that overexpression of EIL1 in the Nr (ethylene receptor)
mutant can only partially restore the expression of ripening genes, suggesting
different EILs may play specific roles in gene regulation (Chen et al, 2004).
We have also characterized LeCTR2, which has similarity to CTR1 and also to
EDR1, a CTR1-like Arabidopsis protein involved in defence and stress responses.
Protein-protein interactions between LeCTR2 and six tomato ethylene receptors
indicated that LeCTR2 interacts preferentially with the subfamily I ETR1-type
ethylene receptors LeETR1 and LeETR2, but not the NR receptor, or the subfamily
II receptors LeETR4, LeETR5 and LeETR6. The C-terminus of LeCTR2 possesses serine/threonine
kinase activity and is capable of auto-phosphorylation and phosphorylation of
myelin basic protein in vitro. Overexpression of the LeCTR2 N-terminus in tomato
resulted in altered growth habit, including reduced stature, loss of apical
dominance, highly-branched inflorescences and fruit trusses, indeterminate shoots
in place of determinate flowers, and prolific adventitious shoot development
from the rachis or rachillae of the leaves. Expression of the ethylene-responsive
genes E4 and chitinase B was up-regulated in transgenic plants, but ethylene
production and the level of mRNA for the ethylene biosynthetic gene ACO1 was
unaffected. Leaves and fruit of transgenic plants also displayed enhanced susceptibility
to infection by the fungal pathogen Botrytis cinerea, compared to the wild type
The results suggest that LeCTR2 plays a role in ethylene signalling, development
and defence, probably through its interactions with the ETR1-type ethylene receptors
of subfamily I (see Lin et al., 2008b).
Additional signalling components that regulate the ethylene receptors have now
been identified by mutant analysis in Arabidopsis (such as RTE1, RAN1), and
we have identified a novel receptor-binding protein in tomato (Lin et al, 2008c).
SlTPR1* encodes a tetratricopeptide repeat (TPR) protein isolated by interaction
with the tomato ethylene receptor NR in a yeast two-hybrid screen. Previously
identified TPR proteins from other organisms are involved in cell signalling
and regulation through protein-protein interactions. Overexpression of SlTPR1
in tomato resulted in pleiotropic effects on plant growth habit, including reduced
stature, delayed, deformed and infertile flowers, epinasty, reduced apical dominance,
and altered leaf morphology. These studies indicate that SlTPR1 functions as
a positive regulator in ethylene signalling and its persistent overexpression
alters a range of developmental events, including some auxin responses. This
led us to propose models in which SlTPR1 modulates ethylene receptor levels
or the ethylene signalling response (Lin et al., 2008c). We have also shown
that a related protein in Arabidopsis, AtTRP1, binds to the ERS (NR-type) receptor
in a yeast 2-hybrid system, and shown by in vivo co-immunoprecipitation that
it binds to ERS1 dimers in cell plant membranes (Lin, Ho and Grierson, unpublished).
These results suggest that far from indicating functional redundancy, the multiple
ethylene signalling components actually participate in a more subtle and complex
process of regulation than previously recognised.

*Since the tomato taxonomic name was changed from Lycopersicon esculentum to
Solanum Lycopersicon, new tomato genes are prefixed by Sl.

GRIMWADE, J.A., GRIERSON, D. & WHITTINGTON, W.J. The
effect of differences in time to maturity on the quality of seed produced
by different varieties of sugar beet. Seed Sci. Technol. 15, 135 145, 1987.

GRIERSON, D., SLATER, A., SPEIRS, J. & TUCKER, G.A.
The appearance of polygalacturonase mRNA in tomatoes: one of a series of
changes in gene expression during development and ripening. Planta 163,
263 271, 1985.